Angiogenesis is the process of forming new capillaries from preexisting blood vessels and is an essential component of embryogenesis, normal physiological growth, repair, and tumor expansion. Although a variety of factors can modulate endothelial cell (EC) responses in vitro and blood vessel growth in vivo, only vascular endothelial growth factor (VEGF) family members and the angiopoietins are believed to act almost exclusively on vascular ECs. Yancopoulos et al., Nature 407:242-48 (2000).
The angiopoietins were discovered as ligands for the Ties, a family of tyrosine kinases that is selectively expressed within the vascular endothelium. Yancopoulos et al., Nature 407:242-48 (2000). There are now four definitive members of the angiopoietin family. Angiopoietin-3 and -4 (Ang-3 and Ang-4) may represent widely diverged counterparts of the same gene locus in mouse and man. Kim et al., FEBS Let, 443:353-56 (1999); Kim et al., J Biol Chem 274:26523-28 (1999). Ang-1 and Ang-2 were originally identified in tissue culture experiments as agonist and antagonist, respectively. Davis et al., Cell 87:1161-69 (1996); Maisonpierre et al., Science 277:55-60 (1997). All of the known angiopoietins bind primarily to Tie2, and both Ang-1 and -2 bind to Tie2 with an affinity of 3 nM (Kd). Maisonpierre et al., Science 277:55-60 (1997). Ang-1 was shown to support EC survival and to promote endothelium integrity, Davis et al., Cell 87:1161-69 (1996); Kwak et al., FEBS Lett 448:249-53 (1999); Suri et al., Science 282:468-71 (1998); Thurston et al., Science 286: 2511-14 (1999); Thurston et al., Nat. Med. 6:460-63 (2000), whereas Ang-2 had the opposite effect and promoted blood vessel destabilization and regression in the absence of the survival factors VEGF or basic fibroblast growth factor. Maisonpierre et al., Science 277:55-60 (1997). However, many studies of Ang-2 function have suggested a more complex situation. Ang-2 might be a complex regulator of vascular remodeling that plays a role in both vessel sprouting and vessel regression. Supporting such roles for Ang-2, expression analyses reveal that Ang-2 is rapidly induced, together with VEGF, in adult settings of angiogenic sprouting, whereas Ang-2 is induced in the absence of VEGF in settings of vascular regression. Holash et al., Science 284:1994-98 (1999); Holash et al., Oncogene 18:5356-62 (1999). Consistent with a context-dependent role, Ang-2 binds to the same endothelial-specific receptor, Tie-2, which is activated by Ang-1, but has context-dependent effects on its activation. Maisonpierre et al., Science 277:55-60 (1997).
Corneal angiogenesis assays have shown that both Ang-1 and Ang-2 had similar effects, acting synergistically with VEGF to promote growth of new blood vessels. Asahara et al., Circ. Res. 83:233-40 (1998). The possibility that there was a dose-dependent endothelial response was raised by the observation that in vitro at high concentration, Ang-2 can also be pro-angiogenic. Kim et al., Oncogene 19:4549-52 (2000). At high concentration, Ang-2 acts as an apoptosis survival factor for endothelial cells during serum deprivation apoptosis through activation of Tie2 via PI-3 kinase and Akt pathway. Kim et al., Oncogene 19:4549-52 (2000).
Other in vitro experiments suggested that during sustained exposure, the effects of Ang-2 may progressively shift from that of an antagonist to an agonist of Tie2, and at later time points, it may contribute directly to vascular tube formation and neovessel stabilization. Teichert-Kuliszewska et al., Cardiovasc. Res. 49:659-70 (2001). Furthermore, if ECs were cultivated on fibrin gel, activation of Tie2 with Ang-2 was also observed, perhaps suggesting that the action of Ang-2 could depend on EC differentiation state. Teichert-Kuliszewska et al., Cardiovasc. Res. 49:659-70 (2001). In microvascular EC cultured in a three-dimensional collagen gel, Ang-2 can also induce Tie2 activation and promote formation of capillary-like structures. Mochizuki et al., J. Cell. Sci. 115:175-83 (2002). Use of a 3-D spheroidal coculture as an in vitro model of vessel maturation demonstrated that direct contact between ECs and mesenchymal cells abrogates responsiveness to VEGF, whereas the presence of VEGF and Ang-2 induced sprouting. Korff et al., Faseb J. 15:447-57 (2001). Etoh et al. demonstrated that ECs that constitutively express Tie2, the expression of MMP-1, -9 and u-PA were strongly up-regulated by Ang-2 in the presence of VEGF. Etoh, et al., Cancer Res. 61:2145-53 (2001). With an in vivo pupillary membrane model, Lobov et al. showed that Ang-2 in the presence of endogenous VEGF promotes a rapid increase in capillary diameter, remodeling of the basal lamina, proliferation and migration of endothelial cells, and stimulates sprouting of new blood vessels. Lobov et al., Proc. Natl. Acad. Sci. USA 99:11205-10 (2002). By contrast, Ang-2 promotes endothelial cell death and vessel regression without endogenous VEGF. Lobov et al., Proc. Natl. Acad. Sci. USA 99:11205-10 (2002). Similarly, with an in vivo tumor model, Vajkoczy et al. demonstrated that multicellular aggregates initiate vascular growth by angiogenic sprouting via the simultaneous expression of VEGFR-2 and Ang-2 by host and tumor endothelium. Vajkoczy et al., J. Clin. Invest. 109:777-85 (2002). This model illustrated that the established microvasculature of growing tumors is characterized by a continuous remodeling, putatively mediated by the expression of VEGF and Ang-2. Vajkoczy et al., J. Clin. Invest. 109:777-85 (2002).
Knock-out mouse studies of Tie-2 and Angiopoietin-1 show similar phenotypes and suggest that Angiopoietin-1 stimulated Tie-2 phosphorylation mediates remodeling and stabilization of developing vessel, promoting blood vessel maturation during angiogenesis and maintenance of endothelial cell-support cell adhesion (Dumont et al., Genes & Development, 8:1897-1909 (1994); Sato, Nature, 376:70-74 (1995); (Thurston, G. et al., 2000 Nature Medicine: 6, 460-463)). The role of Angiopoietin-1 is thought to be conserved in the adult, where it is expressed widely and constitutively (Hanahan, Science, 277:48-50 (1997); Zagzag, et al., Exp Neurology, 159:391-400 (1999)). In contrast, Angiopoietin-2 expression is primarily limited to sites of vascular remodeling where it is thought to block the constitutive stabilizing or maturing function of Angiopoietin-1, allowing vessels to revert to, and remain in, a plastic state which may be more responsive to sprouting signals (Hanahan, 1997; Holash et al., Oncogene 18:5356-62 (1999); Maisonpierre, 1997). Studies of Angiopoietin-2 expression in pathological angiogenesis have found many tumor types to show vascular Angiopoietin-2 expression (Maisonpierre et al., Science 277:55-60 (1997)). Functional studies suggest Angiopoietin-2 is involved in tumor angiogenesis and associate Angiopoietin-2 overexpression with increased tumor growth in a mouse xenograft model (Ahmad, et al., Cancer Res., 61:1255-1259 (2001)). Other studies have associated Angiopoietin-2 overexpression with tumor hypervascularity (Etoh, et al., Cancer Res. 61:2145-53 (2001); Tanaka et al., Cancer Res. 62:7124-29 (2002)).
In recent years Angiopoietin-1, Angiopoietin-2 and/or Tie-2 have been proposed as possible anti-cancer therapeutic targets. For example U.S. Pat. No. 6,166,185, U.S. Pat. No. 5,650,490 and U.S. Pat. No. 5,814,464 each disclose anti-Tie-2 ligand and receptor antibodies. Studies using soluble Tie-2 were reported to decrease the number and size of tumors in rodents (Lin, 1997; Lin 1998). Siemester et al. (1999) generated human melanoma cell lines expressing the extracellular domain of Tie-2, injected these into nude mice and reported soluble Tie-2 to result in significant inhibition of tumor growth and tumor angiogenesis. Given both Angiopoietin-1 and Angiopoietin-2 bind to Tie-2, it is unclear from these studies whether Angiopoietin-1, Angiopoietin-2 or Tie-2 would be an attractive target for anti-cancer therapy. However, effective anti-Angiopoietin-2 therapy is thought to be of benefit in treating diseases such as cancer, in which progression is dependant on aberrant angiogenesis where blocking the process can lead to prevention of disease advancement (Folkman, J., Nature Medicine. 1: 27-31 (1995). In addition some groups have reported the use of antibodies that bind to Angiopoietin-2, See, for example, U.S. Pat. No. 6,166,185 and U.S. Patent Application Publication No. 2003/0124129 A1. Study of the effect of focal expression of Angiopoietin-2 has shown that antagonizing the Angiopoietin-1/Tie-2 signal loosens the tight vascular structure thereby exposing ECs to activating signals from angiogenesis inducers, e.g. VEGF (Hanahan, 1997). This pro-angiogenic effect resulting from inhibition of Angiopoietin-1 indicates that anti-Angiopoietin-1 therapy would not be an effective anti-cancer treatment.
Ang-2 is expressed during development at sites where blood vessel remodeling is occurring. Maisonpierre et al., Science 277:55-60 (1997). In adult individuals, Ang-2 expression is restricted to sites of vascular remodeling as well as in highly vascularized tumors, including glioma, Osada et al., Int. J. Oncol. 18:305-09 (2001); Koga et al., Cancer Res. 61:6248-54 (2001), hepatocellular carcinoma, Tanaka et al, J. Clin. Invest. 103:341-45 (1999), gastric carcinoma, Etoh, et al., Cancer Res. 61:2145-53 (2001); Lee et al, Int. J. Oncol. 18:355-61 (2001), thyroid tumor, Bunone et al., Am J Pathol 155:1967-76 (1999), non-small cell lung cancer, Wong et al., Lung Cancer 29:11-22 (2000), and cancer of colon, Ahmad et al., Cancer 92:1138-43 (2001), and prostate Wurmbach et al., Anticancer Res. 20:5217-20 (2000). Some tumor cells are found to express Ang-2. For example, Tanaka et al., J. Clin. Invest. 103:341-45 (1999) detected Ang-2 mRNA in 10 out of 12 specimens of human hepatocellular carcinoma (HCC). Ellis' group reported that Ang-2 is expressed ubiquitously in tumor epithelium. Ahmad et al., Cancer 92:1138-43 (2001). Other investigators reported similar findings. Chen et al., J. Tongji Med. Univ. 21:228-30, 235 (2001). By detecting Ang-2 mRNA levels in archived human breast cancer specimens, Sfilogoi et al., Int. J. Cancer 103:466-74 (2003) reported that Ang-2 mRNA is significantly associated with auxiliary lymph node invasion, short disease-free time and poor overall survival. Tanaka et al., Cancer Res. 62:7124-29 (2002) reviewed a total of 236 patients of non-small cell lung cancer (NSCLC) with pathological stage-I to -IIIA, respectively. Using immunohistochemistry, they found that 16.9% of the NSCLC patients were Ang-2 positive. The microvessel density for Ang-2 positive tumor is significantly higher than that of Ang-2 negative. Such an angiogenic effect of Ang-2 was seen only when VEGF expression was high. Moreover, positive expression of Ang-2 was a significant factor to predict a poor postoperative survival. Tanaka et al., Cancer Res. 62:7124-29 (2002). However, they found no significant correlation between Ang-1 expression and the microvessel density. Tanaka et al., Cancer Res. 62:7124-29 (2002). These results suggest that Ang-2 is an indicator of poor prognosis patients with several types of cancer.
Recently, using an Ang-2 knockout mouse model, Yancopoulos' group reported that Ang-2 is required for postnatal angiogenesis. Gale et al., Dev. Cell 3:411-23 (2002). They showed that the developmentally programmed regression of the hyaloid vasculature in the eye does not occur in the Ang-2−/− mice and their retinal blood vessels fail to sprout out from the central retinal artery. Gale et al., Dev. Cell 3:411-23 (2002). They also found that deletion of Ang-2 results in profound defects in the patterning and function of the lymphatic vasculature. Gale et al., Dev. Cell 3:411-23 (2002). Genetic rescue with Ang-1 corrects the lymphatic, but not the angiogenesis defects. Gale et al., Dev. Cell 3:411-23 (2002).
Peters and his colleagues reported that soluble Tie2, when delivered either as recombinant protein or in a viral expression vector, inhibited in vivo growth of murine mammary carcinoma and melanoma in mouse models. Lin et al., Proc. Natl. Acad. Sci. USA 95:8829-34 (1998); Lin et al., J. Clin. Invest. 100:2072-78 (1997). Vascular densities in the tumor tissues so treated were greatly reduced. In addition, soluble Tie2 blocked angiogenesis in the rat corneal stimulated by tumor cell conditioned media. Lin et al., J. Clin. Invest. 100:2072-78 (1997). Furthermore, Isner and his team demonstrated that addition of Ang-2 to VEGF promoted significantly longer and more circumferential neovascularity than VEGF alone. Asahara et al., Circ. Res. 83:233-40 (1998). Excess soluble Tie2 receptor precluded modulation of VEGF-induced neovascularization by Ang-2. Asahara et al., Circ. Res. 83:233-40 (1998). Siemeister et al., Cancer Res. 59:3185-91 (1999) showed with nude mouse xenografts that overexpression of the extracellular ligand-binding domains of either Flt-1 or Tie2 in the xenografts results in significant inhibition of pathway could not be compensated by the other one, suggesting that the VEGF receptor pathway and the Tie2 pathway should be considered as two independent mediators essential for the process of in vivo angiogenesis. Siemeister et al., Cancer Res. 59:3185-91 (1999). This is proven by a more recent publication by White et al., Proc. Natl. Acad. Sci. USA 100:5028-33 (2003). In their study, it was demonstrated that a nuclease-resistant RNA aptamer that specifically binds and inhibits Ang-2 significantly inhibited neovascularization induced by bFGF in the rat corneal micropocket angiogenesis model.